摘要
目的探讨早期自噬激活对醛固酮诱导足细胞损伤的保护作用。方法体外培养永生性小鼠足细胞系，分别在醛固酮刺激6、12、24、48h后应用AnnexinV结合流式细胞术检测足细胞凋亡；透射电子显微镜检测足细胞凋亡小体和自噬小体；Western印迹检测自噬标志蛋白微管相关蛋白1轻链3（LC3）、凋亡相关蛋白caspase-3及足细胞分子nephrin的表达；实时定量PCR检测自噬相关基因Beclin-1的表达。结果醛固酮（10^-7mol／L）呈时间依赖性诱导足细胞凋亡，下调nephrin表达。与对照组比较，醛固酮刺激24h足细胞凋亡增加26．5％（P〈0．05），nephrin表达降低28．0％（P〈0．05）。醛固酮（10^-7mol／L）呈时间依赖性诱导足细胞发生自噬。与对照组比较，醛固酮刺激6h自噬相关基因Beclin-1表达显著上调（P〈0．05）；醛固酮刺激12h自噬标志蛋白LC3-Ⅱ／LC3-Ⅰ比值增加（P〈0．05）。应用3-甲基腺嘌呤（3-MA）抑制自噬后，醛固酮诱导足细凋亡的时间提前至刺激后12h。3-MA处理组与醛固酮刺激24h比较，足细胞凋亡增加39．0％（P〈0．05），nephrin表达进一步下调19．5％（P〈0．05），并且caspase-3明显活化（P〈0．05）。结论醛固酮可诱导足细胞发生自噬和凋亡；醛固酮刺激12h时自噬明显活化；刺激24h时凋亡显著增加。早期自噬激活可抑制醛固酮诱导的足细胞凋亡，并减轻足细胞损伤。以足细胞自噬为靶点可能成为治疗肾小球疾病的新研究方向。
Objective To explore the protection of early autophagy activation on podocyte injury induced by aldosterone. Methods In vitro cultured mouse podoeyte clones （MPCS） were treated with aldosterone for 6, 12, 24, 48 h respectively. Apoptosis of podocytes was detected by Annexin V combined with flow cytometry. After 24 h treatment with aldosterone, the existence of apoptotic body and autophagosome was observed by electron microscopy. The protein expressions of LC3, caspase- 3 and nephrin were examined by Western blotting. The mRNA expression of Beclin-1 was detected by real-time PCR. Results The induction of apoptosis and autophagy by aldosterone in podocytes was in time- dependent mannner. After 24 h treatment with aldosterone, the apoptosis was increased by 26.5% （P 〈 0.05） and the expression of nephrin was decreased by 28.0% （P 〈 0.05） compared to control group. Aldosterone remarkably induced the expression of Beclin- 1 at 6 h and promoted the transformation of LC3-Ⅰ to LC3- Ⅱ at 12 h （P 〈 0.05）. Compared to simple aldosterone treatment, the apoptosis rate of podocyte was increased by 39.0% （P 〈 0.05 ）and the expression of nephrin was declined by 19.5% （P 〈 0.05） after 3-methyladenine （3- MA） pre- treatment. Conclusions Aldosterone can induce autophagy and apoptosis in podocytes. Autophagy occurs earlier （12 h） than apoptosis （24 h）. The occurrence of autophagy can inhibit the apoptosis, so the autophagy pathway may be a new research topic of glomerular disease treatment.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2012年第11期835-839,共5页
Chinese Journal of Nephrology
基金
国家自然科学基金（30971376）
江苏省自然科学基金（BK2009046）
关键词
足细胞
醛固酮
凋亡
自噬
Podocytes
Aldosterone
Apoptosis
Autophagy
